Introduction

Apoptosis is an irreversible self-destruct program triggered by a variety of physiological and stress stimuli. It is important for cell development and tissue homeostasis [22]. Morphological changes associated with apoptosis include membrane blebbing, cell shrinkage, nuclear and cytoplasmic condensation, and intranucleosomal DNA cleavage [10]. Apoptosis is primarily mediated by caspases [13, 19], which are cysteine proteases with aspartate specificity that are activated by the cleavage of inactive zymogens (procaspases). Caspase-3 is one of the key downstream members of the aspartate-specific cysteine protease family that is thought to be an essential effector of cell death [7, 17]. Its activation requires proteolytic cleavage of the inactive procaspase-3 into 17–20-kDa and 12-kDa subunits. Activated caspase-3 is, in turn, responsible for the proteolytic cleavage of many key proteins, such as the nuclear poly (ADP-ribose) polymerase (PARP) that is involved in DNA repair. PARP cleavage to an 85-kDa fragment is a crucial event in the commitment to undergo apoptosis [21].

Chlamydiae, which are obligate intracellular bacterial pathogens, can persist in the infected host cells for long periods of time. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli by inhibition of caspase-3 activation and blockade of mitochondrial cytochrome c release [4]. However, it is not clear whether chlamydial infection directly inhibits caspase-3 activation or blocks upstream steps of apoptosis. Signal transduction pathways are linked to the apoptotic machinery, for example, activation of the c-Jun N-terminal kinase and the p38 mitogen-activated protein kinase protein cascades is associated with increased expression of proapoptotic proteins [9, 11]. On the contrary, activation of the Raf/MEK/ERK pathway is associated with increased expression of antiapoptotic proteins [20, 25]. Perkins et al. [14, 15] reported herpes simplex virus type 2 antiapoptotic activity is Raf dependent and involves activation of MEK/ERK. Chlamydiae are the same as virus that depend on host cells for their replication. In addition, chlamydiae can also modulate host cells signaling pathways [1, 8, 27]. We wanted to know whether activation of Raf/MEK/ERK pathway is involved in chlamydial antiapoptotic activity. In the present study, we tested our hypothesis by evaluating whether chlamydial antiapoptotic activity is inhibited by MEK-specific chemical inhibitor U0126 and Raf-specific chemical inhibitors GW5074. We found the percentage of apoptosis induced by STS was significantly higher in Chlamydia-infected cells treated with U0126 or GW5074 than untreated cells. In addition, procaspase-3 and PARP were cleft and DNA fragmentation was seen in Chlamydia-infected cells treated with U0126 or GW5074. The result indicates that the Raf/MEK/ERK survival pathway is involved in chlamydial antiapoptotic activity.

Materials and Methods

Cell Culture and Propagation of Chlamydia

HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) with 10% fetal calf serum at 37°C in an incubator supplied with 5% CO2. Chlamydia trachomatis serovar L2 organisms (kindly provided by Dr. Guangming Zhong from the University of Texas Health Science Center) were directly inoculated onto the cell monolayers, 2 h after infection the inoculum was removed and replaced with DMEM with 10% fetal calf serum and 2 μg/ml of cycloheximide (Invitrogen). The cells were collected 48 h later and suspended with SPG (219 mM sucrose, 12 mM phosphate buffer, 5 mM glutamic acid, pH 7.2), and then the cells were briefly sonicated to release the chlamydial elementary bodies (EBs). After centrifugation at 1000×g for 10 min, the supernatant containing EBs was collected, aliquoted and stored at −70°C until use.

Apoptosis Induction

HeLa cells with or without chlamydial infection in 6-well plates or in tissue culture flasks were treated with 1 μM STS (sigma) for 5 h in DMEM with 5% fetal calf serum. The control cells mock-treated with dimethyl sulfoxide (sigma) (diluent for STS) were studied in parallel.

Hoechst Staining

HeLa cells were fixed with 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS) for 20 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for an additional 4 min. The cells were stained with the fluorescent DNA-binding dye Hoechst 32258 (Sigma) for 30 min at room temperature. The condensed or fragmented apoptotic nuclei were observed under fluorescence microscope.

DNA Ladder Assay

HeLa cells with or without chlamydial infection and apoptosis induction were collected by trypsinization and centrifuged at 1000×g for 10 min in PBS. The cells were lysed in a lysis buffer containing 10 mM Tris, pH 7.6, 10 mM EDTA, 50 mM NaCl, and 0.5% SDS at 65°C for 2 h. After extraction with a mixture of phenol, chloroform, and isoamyl alcohol, DNA was precipitated by sodium acetate and water-free ethanol and centrifuged at 12000×g for 20 min. The DNA was washed with 75% ethanol twice and dissolved in TE buffer (10 mmol/l Tris–HCl, 1 mmol/l EDTA, pH 8.0) at 37°C for 30 min in the presence of RNase. After quantification with spectrophotometer, 10 μg of DNA was separated on a 3% agarose gel. Gel was stained with 0.1 μg of ethidium bromide per ml and visualized by exposure to UV light.

Western Blot Assay

HeLa cells with or without chlamydial infection and apoptosis induction were lysed with lysis buffer (0.5% NP-40; 0.1% SDS; 10 mM DTT) in the presence of protease inhibitor cocktail (Amresco) for 30 min on ice, after centrifugation at 12000×g at 4°C for 10 min, the proteins were separated by SDS–PAGE and then transferred to nitrocellulose membranes. The membranes were incubated in TN-T buffer (0.5 M Tris–HCl, pH 7.4, 1.5 M NaCl, 0.1% Tween) containing 5% nonfat milk to block nonspecific binding, and then primary antibodies were applied. These include mouse monoclonal antibodies to ERK1/2, pERK1/2, Raf-1, Tyr340-phosphorylated Raf-1(Cell Signaling Technology), and PARP (Roche Molecular Biochemicals). Primary antibody binding was probed with a horseradish peroxidase-conjugated secondary antibody and visualized with an ECL kit (Pierce).

Caspase-3 Activity Assay

Caspase-3 activity was measured using a caspase-3 activity kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. In brief, HeLa cells were collected by trypsinization, after washing with cold PBS twice, the cell samples were resuspended in lysis buffer for 20 min on ice. The lysate was centrifuged at 16000×g at 4°C for 15 min. Caspase-3 activity assays were performed in 96-well microtitre plates by incubation of 10 μl cell lysate protein in 80 μl reaction buffer containing 10 μl caspase-3 substrate (Ac-DEVD-pNA) at 37°C for 2 h. Samples were measured with an ELISA reader at an absorbance of 405 nm. Caspase-3 activities were expressed as the percentage of enzyme activity compared to control. All experiments were carried out in triplicate.

Statistical Analyses

The statistical software SPSS version 13.0 was used for data analyses. All data were expressed as mean ± standard deviations (SD). A P value of 0.05 or less was considered as statistically significant.

Result

Chlamydial Infection can Activate Raf/MEK/ERK Pathway

Chlamydial infection can alter the profile of host transcription genes at a broad range [23]. The Raf/MEK/ERK pathway is linked to the antiapoptotic machinery. We investigated whether Raf/MEK/ERK pathway is activated in Chlamydia-infected cells. As shown in Fig. 1a, the cells infected with chlamydiae for 12 h displayed a higher level of phosphorylated ERK1/2 and Raf-1, and the levels of phosphorylated ERK1/2 and Raf-1 continued to increase as the infection progressed. The levels of total ERK1/2 and Raf-1 were similar in all cultures. Because MEK1/2 are the direct upstream kinases of ERK1/2, we used the MEK1/2-specific inhibitor U0126 to test whether ERK1/2 are activated by phosphorylated MEK1/2. As expected (Fig. 1b), U0126 effectively inhibited ERK1/2 phosphorylation in a dose-dependent manner, which demonstrate MEK1/2 are also activated in Chlamydia-infected cells.

Fig. 1
figure 1

Chlamydial infection can activate Raf/MEK/ERK pathway. a Activation of ERK1/2 and Raf during chlamydial infection. HeLa cells with or without chlamydial infection were harvested at the times postinfection indicated at the top of the figure for Western Blot detection of pERK1/2 (top panel) and pRaf (third panel), total ERK1/2 (second panel) and Raf (bottom panel). b The effect of MEK inhibition on ERK1/2 activation. HeLa cells with or without chlamydial infection and in the presence or absence of the various concentrations of the MEK inhibitor U0126 indicated at the top of the figure were harvested 36 h after infection for Western Blot detection of pERK1/2 (top panel) and total ERK1/2 (bottom panel)

The Percentage of Apoptotic Cells Induced by STS is Significantly Higher in Chlamydia-Infected Cells Treated with U0126 or GW5074 Than Untreated Cells

Having shown that Raf/MEK/ERK pathway is activated in Chlamydia-infected cells, we wanted to know whether chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK pathway. HeLa cells with or without chlamydial infection were treated with STS in the presence or absence of MEK inhibitor U0126 or Raf inhibitor GW5074 and examined for apoptosis by Hoechst staining. As show in Fig. 2, a dose-dependent increase in the percentage of apoptotic cells induced by STS was seen in Chlamydia-infected cells treated with U0126 (32 ± 2.2)%, (63 ± 3.1)%, and (89 ± 3.4) % for 10, 20, and 50 μM U0126, respectively, compared to similarly infected but untreated cells (2.26 ± 1.3)% (P < 0.05). Significantly, U0126 treatment had no effect on the survival of uninfected cells. Similar results were obtained in infected cells treated with GW5074 (data not shown). The data suggest that the chlamydial antiapoptotic activity requires Raf/MEK/ERK activation.

Fig. 2
figure 2

Chlamydial antiapoptotic activity is inhibited in UO126-treated cells. a HeLa cells with or without chlamydial infection and in the presence or absence of MEK inhibitor U0126 were treated with 1 μM STS for 5 h. The cell samples were then stained with Hoechst dye. Hoechst-positive cells were counted in five randomly microscopic fields and the mean percent of apoptotic cells were expressed as means ± SD. The figure shows the results from three independent experiments. HeLa cells with (df) or without (b, c, g) chlamydial infection were treated with (c, e, f) or without (b, d, g) 1 μM STS for 5 h in the presence (f, g) or absence (be) of 50 μM U0126. The cell samples were then stained with Hoechst and viewed under a fluorescence microscope

DNA Fragmentation Induced by STS was Developed in Chlamydia-Infected Cells Treated with U0126 or GW5074

Because DNA fragmentation is a hallmark of apoptosis, we wanted to know whether inhibition of Raf/MEK/ERK pathway can lead to DNA ladder formation induced by STS in Chlamydia-infected cells. HeLa cells with or without infection and apoptosis induction were treated with U0126 or GW5074, and genomic DNA was examined for fragmentation as described in “Materials and Methods”. As shown in Fig. 3, DNA fragmentation induced by STS was seen in U0126 or GW5074-treated cells, but not in untreated cells and cells that were treated only with inhibitor. The data indicate that inhibition of DNA fragmentation induced by STS in Chlamydia-infected cells is dependent on Raf/MEK/ERK survival pathway.

Fig. 3
figure 3

DNA fragmentation is induced in Chlamydia-infected cells treated with STS in the presence of inhibitor U0126 or GW5074. Cell samples were processed as described at the top of the figure. Genomic DNA was extracted at 36 h postinfection and separated on 3% agarose gels as described in “Materials and Methods

Inhibition of Caspase-3 Activation Induced by STS in Chlamydia-Infected Cells Involves Raf/MEK/ERK Survival Pathway

Since caspase-3 has been shown to play a pivotal role in the execution phase of apoptosis [12], we examined caspase-3 activation induced by STS in Chlamydia-infected cells treated with or without U0126 or W5074. As shown in Fig. 4, caspase-3 activation was increased significantly in infected cells treated with U0126 or GW5074, but not in untreated cells and cells that were treated only with inhibitor, which suggest inhibition of caspase-3 activation in Chlamydia-infected cells involves Raf/MEK/ERK survival pathway.

Fig. 4
figure 4

Caspase-3 is activated in Chlamydia-infected cells treated with STS in the presence of inhibitor U0126 or GW5074. Cell samples were processed as described at the bottom of the figure. Caspase-3 activities were determined using a caspase-3 activity kit. Values were expressed as the ratio of caspase-3 activation levels to control levels, and the value of control was set to 1. The relative values of all samples were determined and expressed as means ± SD. The figure shows the results from three independent experiments

PARP is Cleft in Chlamydia-Infected Cells Treated with STS in the Presence of Inhibitor U0126 or GW5074

Because PARP cleavage by caspase-3 is a hallmark of the commitment to undergo apoptosis, we investigated whether PARP was cleft in Chlamydia-infected cells treated with STS in the presence of inhibitor U0126 or GW5074. As shown in Fig. 5, an 85-kDa band consistent with the PARP cleavage product was seen in Chlamydia-infected cells treated with U0126 or GW5074. On the contrary, a 116-kDa band consistent with uncleaved PARP was seen in untreated cells and cells that were treated only with inhibitor. The data indicate that inhibition of PARP cleavage in Chlamydia-infected involves Raf/MEK/ERK survival pathway.

Fig. 5
figure 5

PARP is cleft in Chlamydia-infected cells treated with STS in the presence of inhibitor U0126 or GW5074. HeLa monolayer cells were infected with Chlamydia trachomatis serovar L2 in the presence or absence of inhibitor U0126 or GW5074, the whole cell lysates were collected at 42 h postinfection to test the PARP cleavage product by Western Blot. The 85-kDa band is consistent with the PARP cleavage product. The 116-kDa band is consistent with uncleaved PARP

Discussion

Chlamydiae depend on host cells for their replication, and they need a safe and rich niche to live in from their host cells. To complete the entire infectious cycle, chlamydiae need to obtain nutrients and metabolism materials from host cells by modulating host signaling pathways [2, 6]. Previous studies showed that activation of Raf/MEK/ERK pathway is essential for chlamydial acquisition of host glycerophospholipids in order to maintain growth [18], which inevitably requires chlamydiae to manipulate host cells and to prevent but not to induce host cell apoptosis. Chlamydiae have acquired various strategies to prevent apoptosis, including down-regulation of BH3-only proteins [3, 5, 26], inhibition of Bax and Bak activation [24], blockade of mitochondrial cytochrome c release, and caspase-3 activation [4]. Although chlamydial infection can activate Raf/MEK/ERK pathway, it is unclear whether the survival pathway is involved in the antiapoptotic activity of chlamydiae. In our studies, we have not only shown that chlamydial infection can activate the Raf/MEK/ERK pathway, which was consistent with previous published observations [16, 18], but also demonstrated chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK survival pathway.

The suitability of various assays for the detection of apoptosis has recently come under scrutiny. Our definition of apoptosis is based on a multiplicity of criteria, including nuclear morphology, DNA fragmentation, caspase-3 activation, and PARP cleavage, a crucial factor in the cell’s commitment to undergo apoptosis, with a good degree correlation between the various assays. The current study presented compelling evidence that chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK survival pathway. (a) The levels of phosphorylated Raf, MEK, and ERK1/2 were significantly higher in Chlamydia-infected cells than uninfected cells, and were increased as the infection progressed, (b) A dose-dependent increase in the percentage of apoptotic cells induced by STS was seen in Chlamydia-infected cells treated with various concentrations of MEK-specific inhibitor U0126 compared to similarly infected but untreated cells. Similar results were obtained in GW5074-treated cells infected with chlamydiae (data not shown), (c) DNA fragmentation, a hallmark of apoptosis, induced by STS was seen in Chlamydia-infected cells treated with U0126 or GW5074. However, the infected cells treated only with STS and cells that were treated only with inhibitor did not induce DNA fragmentation, and (d) Procaspase-3 and PARP were cleft in Chlamydia-infected cells treated with STS in the presence U0126 or GW5074. However, the levels of caspase-3 activation and PARP cleavage were not increased in infected cells treated only with STS and cells that were treated only with inhibitor.

Because the Raf/MEK/ERK signaling cascade is involved in the expression of antiapoptotic proteins, we assume that the activation of Raf/MEK/ERK pathway results in increased transcription of antiapoptotic proteins. Indeed, Rajalinga et al. [16] demonstrated Mcl-1 protein was up-regulated in a MAPK-dependent fashion in Chlamydia-infected cells. However, the mechanism of chlamydial antiapoptotic activity is very complex, it is possible that other antiapoptotic proteins are up-regulated in a MAPK-dependent fashion besides Mcl-1 protein. Interestingly, other intracellular pathogens have also acquired the ability to up-regulate antiapoptotic proteins by activating Raf/MEK/ERK pathway. For example, the herpes simplex virus 2 can up-regulate the antiapoptotic protein Bag-1 in a MAPK-dependent fashion, Bag-1 also binds c-Raf and activates its kinase activity, thereby providing a positive feedback loop for the Raf/MEK/ERK survival pathway [14]. Chlamydiae are the same as virus that must depend on host cells for their replication, which prompt that Bag-1 may be un-regulated in a MAPK-dependent fashion in Chlamydia-infected cells.

In conclusion, the present data demonstrate that chlamydial antiapoptotic activity involves activation of Raf/MEK/ERK survival pathway. The activation of Raf/MEK/ERK pathway will not only promote host cells proliferation, but also provide a safe and rich niche for chlamydiae.